1. Field of the Invention
The invention relates to an apparatus and method for measurement of mononuclear cells contained in analytes.
2. Description of the Related Art
Cell therapy such as stem cell transplantation and donor lymphocyte infusion uses stem cells, lymphocytes or the like for cell transplantation into patients. Stem cells and lymphocytes (hereinafter generically referred to as “mononuclear cells”) for use in cell therapy are mainly contained in blood (e.g. peripheral blood and cord blood) and bone marrow. Hereinafter, mononuclear cell-containing fluid for use in cell therapy, such as blood and bone marrow, is referred to as “cell material.”
Collected cell materials undergo many processes (e.g. removal of cells unnecessary for therapy from the cell materials, freezing and thawing of the cell materials, and the like) before they are transplanted into patients. In such cell materials, therefore, cells necessary for therapy can be damaged. As used herein, the term “damaged cells” refers to dead cells that have their cell membrane damaged and are made nonviable by an influence such as “changes over time after the collection,” “poor storage conditions,” “the process of removing unnecessary cells” and ” the process of freezing and thawing the cell material.” Such dead cells of mononuclear cells (hereinafter referred to as dead mononuclear cells) cannot contribute to engraftment success after transplantation, and thus cell materials rich in dead mononuclear cells are improper to cell therapy.
Therefore, a condition that cell materials should satisfy for effective cell therapy is to contain plenty of living cells of mononuclear cells (hereinafter referred to as living mononuclear cells). Thus, it is important to check the viability of mononuclear cells in cell materials before they are used in cell therapy, and therefore, there is a need to identify living mononuclear cells in cell materials.
Japanese Patent Application Laid-Open (JP-A) No. 2002-148261 discloses a method of classifying and counting abnormal cells, in which abnormal cells such as damaged leukocytes with their cell membrane damaged are classified and counted while leukocytes are measured. In this method, a flow cytometer is used to determine the intensities of fluorescence and scattered light with respect to each cell in blood treated with a fluorescent dye so that leukocytes and damaged leukocytes can be identified based on the intensities of the fluorescence and the scattered light. In the specification of the publication, the term “damaged leukocytes” refers to leukocytes whose cell membrane is damaged by an influence such as changes over time after collection and poor storage conditions.
However, the specification of the publication doesn't disclose the identification of living mononuclear cells including stem cells.
A known method of identifying living mononuclear cells uses 7-aminoactinomycin D (7-AAD), fluorescence-labeled CD34 and fluorescence-labeled CD45 (Michael Keeney, Ian Chin-Yee, Karin Weir, Jan Popma, Rakash Nayar, D. Robert Sutherland, “Single platform flow cytometric absolute CD34+cell counts based on the ISHAGE guidelines,” Cytometry Wiley-Liss, Inc., 1998, Volume 34, Issue 2, P. 61-70). The 7-AAD is a fluorescent dye for staining of nucleic acid. With this dye, dead cells are more stained than living cells, so that the living cells can be identified separately from the dead cells based on the difference in the degree of 7-AAD staining. CD34 is an antibody to a stem cell surface antigen, and CD45 is an antibody to a leukocyte cell surface antigen. Thus, living mononuclear cells can be identified using 7-aminoactinomycin D (7-AAD) and CD34 or CD45 at the same time.
However, the above method involves a complicated process including the modification of the stem or leukocyte cell surface antigen through antigen-antibody reaction at the time of preparing test samples, and it takes much time.